Multiple drug resistance gene atrD of aspergillus nidulans

ABSTRACT

The invention provides isolated nucleic acid compounds encoding a multiple drug resistance protein of  Aspergillus nidulans . Vectors and transformed host cells comprising the multiple drug resistance-encoding DNA of  Aspergillus nidulans  atrD are also provided. The invention further provides assays which utilize these transformed host cells.

This application is a divisional of, and claims the benefit of priorityof, U.S. application Ser. No. 09/328,320 filed Jun. 8, 1999, now U.S.Pat. No. 6,228,615, which is a divisional of U.S. application Ser. No.08/996,545 filed Dec. 23, 1997, now U.S. Pat. No. 5,928,898, thecontents of each of which are herein incorporated by reference in theirentirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to recombinant DNA technology. In particular, theinvention concerns the cloning of nucleic acid encoding a multiple drugresistance protein of Aspergillus nidulans.

BACKGROUND OF THE INVENTION

Multiple drug resistance (MDR) mediated by the human mdr-1 gene productwas initially recognized during the course of developing regimens forcancer chemotherapy (Fojo et al., 1987, Journal of Clinical Oncology5:1922-1927). A multiple drug resistant cancer cell line exhibitsresistance to high levels of a large variety of cytotoxic compounds.Frequently these cytotoxic compounds will have no common structuralfeatures nor will they interact with a common target within the cell.Resistance to these cytotoxic agents is mediated by an outward directed,ATP-dependent pump encoded by the mdr-1 gene. By this mechanism, toxiclevels of a particular cytotoxic compound are not allowed to accumulatewithin the cell.

MDR-like genes have been identified in a number of divergent organismsincluding numerous bacterial species, the fruit fly Drosophilamelanogaster, Plasmodium falciparum, the yeast Saccharomyces cerevisiae,Caenorhabditis elegans, Leishmania donovanii, marine sponges, the plantArabidopsis thaliana, as well as Homo sapiens. Extensive searches haverevealed several classes of compounds that are able to reverse the MDRphenotype of multiple drug resistant human cancer cell lines renderingthem susceptible to the effects of cytotoxic compounds. These compounds,referred to herein as “MDR inhibitors”, include for example, calciumchannel blockers, antiarrhythmics, antihypertensives, antibiotics,antihistamines, immuno-suppressants, steroid hormones, modifiedsteroids, lipophilic cations, diterpenes, detergents, antidepressants,and antipsychotics (Gottesman and Pastan, 1993, Annual Review ofBiochemistry 62:385-427). Clinical application of human MDR inhibitorsto cancer chemotherapy has become an area of intensive focus forresearch.

On another front, the discovery and development of antifungal compoundsfor specific fungal species has also met with some degree of success.Candida species represent the majority of fungal infections, and screensfor new antifungal compounds have been designed to discover anti-Candidacompounds. During development of antifungal agents, activity hasgenerally been optimized based on activity against Candida albicans. Asa consequence, these anti-Candida compounds frequently do not possessclinically significant activity against other fungal species such asAspergillus nidulans. However, it is interesting to note that at higherconcentrations some anti-Candida compounds are able to kill other fungalspecies such as A. nidulans and A. fumigatus. This type of observationsuggests that the antifungal target(s) of these anti-Candida compoundsis present in A. nidulans as well. Such results indicate that A.nidulans may possess a natural mechanism of resistance that permits themto survive in clinically relevant concentrations of antifungalcompounds. Until the present invention, such a general mechanism ofresistance to antifungal compounds in A. nidulans has remainedundescribed.

SUMMARY OF THE INVENTION

The invention provides, inter alia, isolated nucleic acid molecules thatcomprise nucleic acid encoding a multiple drug resistance protein fromAspergillus nidulans, herein referred to as atrD, vectors encoding atrD,and host cells transformed with these vectors.

In another embodiment, the invention provides a method for determiningthe fungal MDR inhibition activity of a compound which comprises:

a) placing a culture of fungal cells, transformed with a vector capableof expressing atrD, in the presence of:

(i) an antifungal agent to which said fungal cell is resistant, but towhich said fungal cell is sensitive in its untransformed state;

(ii) a compound suspected of possessing fungal MDR inhibition activity;and

b) determining the fungal MDR inhibition activity of said compound bymeasuring the ability of the antifungal agent to inhibit the growth ofsaid fungal cell.

In still another embodiment the present invention relates to strains ofA. nidulans in which the atrD gene is disrupted or otherwise mutatedsuch that the atrD protein is not produced in said strains.

In yet another embodiment, the present invention relates to a method foridentifiying new antifungal compounds comprising the use of atrD genedisruption or gene replacement strains of A. nidulans.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated nucleic acid molecules thatcomprise a nucleic acid sequence encoding atrD. The cDNA (complementarydeoxyribonucleic acid) sequence encoding atrD is provided in theSequence Listing as SEQ ID NO: 1. The amino acid sequence of the proteinencoded by atrD is provided in the Sequence Listing as SEQ ID NO: 2.

Those skilled in the art will recognize that the degenerate nature ofthe genetic code enables one to construct many different nucleic acidsequences that encode the amino acid sequence of SEQ ID NO: 2. The cDNAsequence depicted by SEQ ID NO: 1 is only one of many possibleatrD-encoding sequences. Consequently, the constructions described belowand in the accompanying examples for the preferred nucleic acidmolecules, vectors, and transformants of the invention are illustrativeand are not intended to limit the scope of the invention.

All nucleotide and amino acid abbreviations used in this disclosure arethose accepted by the United States Patent and Trademark Office as setforth in 37 C.F.R. §1.822(b)(1994).

The term “vector” refers to any autonomously replicating or integratingagent, including but not limited to plasmids, cosmids, and viruses(including phage), comprising a nucleic acid molecule to which one ormore additional nucleic acid molecules can be added. Included in thedefinition of “vector” is the term “expression vector”. Vectors are usedeither to amplify and/or to express deoxyribonucleic acid (DNA), eithergenomic or cDNA, or RNA (ribonucleic acid) which encodes atrD, or toamplify DNA or RNA that hybridizes with DNA or RNA encoding atrD.

The term “expression vector” refers to vectors which comprise atranscriptional promoter (hereinafter “promoter”) and other regulatorysequences positioned to drive expression of a DNA segment that encodesatrD. Expression vectors of the present invention are replicable DNAconstructs in which a DNA sequence encoding atrD is operably linked tosuitable control sequences capable of effecting the expression of atrDin a suitable host. Such control sequences include a promoter, anoptional operator sequence to control transcription, a sequence encodingsuitable MRNA ribosomal binding sites, and sequences which controltermination of transcription and translation. DNA regions are operablylinked when they are functionally related to each other. For example, apromoter is operably linked to a DNA coding sequence if it controls thetranscription of the sequence, or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to permittranslation.

The term “MDR inhibition activity” refers to the ability of a compoundto inhibit the MDR activity of a host cell, thereby increasing theantifungal activity of an antifungal compound against said host cell.

In the present invention, atrD may be synthesized by host cellstransformed with vectors that provide for the expression of DNA encodingatrD. The DNA encoding atrD may be the natural sequence or a syntheticsequence or a combination of both (“semi-synthetic sequence”). The invitro or in vivo transcription and translation of these sequencesresults in the production of atrD. Synthetic and semi-syntheticsequences encoding atrD may be constructed by techniques well known inthe art. See Brown et al. (1979) Methods in Enzymology, Academic Press,N.Y., 68:109-151. atrD-encoding DNA, or portions thereof, may begenerated using a conventional DNA synthesizing apparatus such as theApplied Biosystems Model 380A,380B, 394 or 3948 DNA synthesizers(commercially available from Applied Biosystems, Inc., 850 LincolnCenter Drive, Foster City, Calif. 94404).

Owing to the natural degeneracy of the genetic code, the skilled artisanwill recognize that a sizable yet definite number of nucleic acidsequences may be constructed which encode atrD. All such nucleic acidsequences are provided by the present invention. These sequences can beprepared by a variety of methods and, therefore, the invention is notlimited to any particular preparation means. The nucleic acid sequencesof the invention can be produced by a number of procedures, includingDNA synthesis, cDNA cloning, genomic cloning, polymerase chain reaction(PCR) technology, or a combination of these approaches. These and othertechniques are described by Maniatis, et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1989), or Current Protocols inMolecular Biology (F. M. Ausubel et al., 1989 and supplements). Thecontents of both of these references are incorporated herein byreference.

In another aspect, this invention provides the cDNA encoding atrD, whichmay be obtained by synthesizing the desired portion of SEQ ID NO:1 or byfollowing the procedure carried out by Applicants. This procedureinvolved construction of a cosmid genomic DNA library from Aspergillusnidulans strain OC-1, a mutant derived from A42355. This library wasscreened for genes related to MDRs using a homologous probe generated byPCR. Degenerate PCR primers directed towards amplification of DNAsequences encoding highly conserved regions found in the ATP-bindingdomain of several MDR genes were synthesized. PCR using these primersand Aspergillus nidulans genomic DNA as template produced anapproximately 400 base pair DNA fragment. The DNA sequence of thisfragment was highly homologous to the ATP-binding region of several MDRsas predicted. This fragment was used as a hybridization probe toidentify cosmid clones containing the entire atrD gene. A subclone fromone such cosmid containing the entire atrD gene was sequenced toascertain the entire sequence of atrD.

To effect the translation of atrD-encoding mRNA, one inserts thenatural, synthetic, or semi-synthetic atrD-encoding DNA sequence intoany of a large number of appropriate expression vectors through the useof appropriate restriction endonucleases and DNA ligases. Synthetic andsemi-synthetic atrD-encoding DNA sequences can be designed, and naturalatrD-encoding nucleic acid can be modified, to possess restrictionendonuclease cleavage sites to facilitate isolation from and integrationinto these vectors. Particular restriction endonucleases employed willbe dictated by the restriction endonuclease cleavage pattern of theexpression vector utilized. Restriction enzyme sites are chosen so as toproperly orient the atrD-encoding DNA with the control sequences toachieve proper in-frame transcription and translation of the atrDmolecule. The atrD-encoding DNA must be positioned so as to be in properreading frame with the promoter and ribosome binding site of theexpression vector, both of which are functional in the host cell inwhich atrD is to be expressed.

Expression of atrD in fungal cells, such as Saccharomyces cerevisiae ispreferred. Suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD(ATCC 53231) and described in U.S. Pat. No. 4,935,350, Jun. 19, 1990) orother glycolytic enzymes such as enolase (found on plasmid pAC1 (ATCC39532)), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmidpHcGAPC1 (ATCC 57090, 57091)), hexokinase, pyruvate decarboxylase,phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglyceratemutase, pyruvate kinase, triosephosphate isomerase, phosphoglucoseisomerase, and glucokinase. Inducible yeast promoters have theadditional advantage of transcription controlled by growth conditions.Such promoters include the promoter regions for alcohol dehydrogenase 2,isocytochrome C, acid phosphotase, degradative enzymes associated withnitrogen metabolism, metallothionein (contained on plasmid vectorpCL28XhoLHBPV (ATCC 39475), U.S. Pat. No. 4,840,896), glyceraldehyde3-phosphate dehydrogenase, and enzymes responsible for maltose andgalactose utilization (GAL1 found on plasmid pRY121 (ATCC 37658) and onplasmid pPST5, described below). Suitable vectors and promoters for usein yeast expression are further described by R. Hitzeman et al., inEuropean Patent Publication No. 73,657A. Yeast enhancers such as the UASGal enhancer from Saccharomyces cerevisiae (found in conjunction withthe CYCI promoter on plasmid YEpsec—hI1beta, ATCC 67024), also areadvantageously used with yeast promoters.

A variety of expression vectors useful in the present invention are wellknown in the art. For expression in Saccharomyces, the plasmid YRp7, forexample, (ATCC-40053, Stinchcomb et al., 1979, Nature 282:39; Kingsmanet al., 1979, Gene 7:141; Tschemper et al., 1980, Gene 10:157) iscommonly used. This plasmid contains the trp gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC 44076 or PEP4-1 (Jones, 1977,Genetics 85:12).

Expression vectors useful in the expression of atrD can be constructedby a number of methods. For example, the cDNA sequence encoding atrD canbe synthesized using DNA synthesis techniques such as those describedabove. Such synthetic DNA can be synthesized to contain cohesive endsthat allow facile cloning into an appropriately digested expressionvector. For example, the cDNA encoding atrD can be synthesized tocontain NotI cohesive ends. Such a synthetic DNA fragment can be ligatedinto a NotI-digested expression vector such as pYES-2 (Invitrogen Corp.,San Diego Calif. 92121).

An expression vector can also be constructed in the following manner.Logarithmic phase Aspergillus nidulans cells are disrupted by grindingunder liquid nitrogen according to the procedure of Minuth et al., 1982(Current Genetics 5:227-231). Aspergillus nidulans mRNA is preferablyisolated from the disrupted cells using the QuickPrep® mRNA PurificationKit (Pharmacia Biotech) according to the instructions of themanufacturer. cDNA is produced from the isolated mRNA using theTimeSaver® cDNA Synthesis Kit (Pharmacia Biotech) using oligo (dT)according to the procedure described by the manufacturer. In thisprocess an EcoRI/NotI adapter (Stratagene, Inc.) is ligated to each endof the double stranded cDNA. The adapter modified cDNA is ligated intothe vector Lambda Zap^(R)II® using the Predigested LambdaZap^(R)II®/EcoRI/CIAP Cloning Kit (Stratagene, Inc.) according to theinstructions of the manufacturer to create a cDNA library.

The library is screened for full-length cDNA encoding atrD using a³²P-radiolabeled fragment of the atrD gene. In this manner, afull-length cDNA clone is recovered from the Aspergillus nidulans cDNAlibrary. A full-length cDNA clone recovered from the library is removedfrom the Lambda Zap^(R)II® vector by digestion with the restrictionendonuclease NotI which produces a DNA fragment encoding atrD. The atrDencoding fragment is subcloned into plasmid pYES2 for expressionstudies. In this plasmid the atrD gene is operably linked to theSaccharomyces cerevisiae GAL1 promoter at the 5′ end, and the yeast cyc1transcription terminator at the 3′ end. This plasmid further comprisesthe ColE1 origin of replication (ColE1) which allows replication inEscherichia coli host cells, and the ampicillin resistance gene (Amp)for selection of E. coli cells transformed with the plasmid grown in thepresence of ampicillin. The expression plasmid further comprises theyeast 2μ origin of replication (2μ ori) allowing replication in yeasthost cells, the yeast URA3 gene for selection of S. cerevisiae cellstransformed with the plasmid grown in a medium lacking uracil, and theorigin of replication from the f1 filamentous phage.

In a preferred embodiment of the invention Saccharomyces cerevisiaeINVSc1 or INVSc2 cells (Invitrogen Corp., Sorrento Valley Blvd., SanDiego Calif. 92121) are employed as host cells, but numerous other celllines are available for this use. The transformed host cells are platedon an appropriate medium under selective pressure (minimal mediumlacking uracil). The cultures are then incubated for a time andtemperature appropriate to the host cell line employed.

The techniques involved in the transformation of yeast cells such asSaccharomyces cerevisiae cells are well known in the art and may befound in such general references as Ausubel et al., Current Protocols inMolecular Biology (1989), John Wiley & Sons, New York, N.Y. andsupplements. The precise conditions under which the transformed yeastcells are cultured is dependent upon the nature of the yeast host cellline and the vectors employed.

Nucleic acid, either RNA or DNA, which encodes atrD, or a portionthereof, is also useful in producing nucleic acid molecules useful indiagnostic assays for the detection of atrD MRNA, atrD cDNA, or atrDgenomic DNA. Further, nucleic acid, either RNA or DNA, which does notencode atrD, but which nonetheless is capable of hybridizing withatrD-encoding DNA or RNA is also useful in such diagnostic assays. Thesenucleic acid molecules may be covalently labeled by known methods with adetectable moiety such as a fluorescent group, a radioactive atom or achemiluminescent group. The labeled nucleic acid is then used inconventional hybridization assays, such as Southern or Northernhybridization assays, or polymerase chain reaction assays (PCR), toidentify hybridizing DNA, cDNA, or RNA molecules. PCR assays may also beperformed using unlabeled nucleic acid molecules. Such assays may beemployed to identify atrD vectors and transformants and in in vitrodiagnosis to detect atrD-like mRNA, CDNA, or genomic DNA from otherorganisms.

U.S. patent application Ser. No. 08/111,680 (now abandoned), the entirecontents of which are hereby incorporated herein by reference, describesthe use of combination therapy involving an antifungal agent possessinga proven spectrum of activity, with a fungal MDR inhibitor to treatfungal infections. This combination therapy approach enables anextension of the spectrum of antifungal activity for a given antifungalcompound which previously had only demonstrated limited clinicallyrelevant antifungal activity. Similarly, compounds with demonstratedantifungal activity can also be potentiated by a fungal MDR inhibitorsuch that the antifungal activity of these compounds is extended topreviously resistant species. To identify compounds useful in suchcombination therapy the present invention provides an assay method foridentifying compounds with Aspergillus nidulans MDR inhibition activity.Host cells that express atrD provide an excellent means for theidentification of compounds useful as inhibitors of Aspergillus nidulansMDR activity. Generally, the assay utilizes a culture of a yeast celltransformed with a vector which provides expression of atrD. Theexpression of atrD by the host cell enables the host cell to grow in thepresence of an antifungal compound to which the yeast cell is sensitiveto in the untransformed state. Thus, the transformed yeast cell cultureis grown in the presence of i) an antifungal agent to which theuntransformed yeast cell is sensitive, but to which the transformed hostcell is resistant, and ii) a compound that is suspected of being an MDRinhibitor. The effect of the suspected MDR inhibitor is measured bytesting for the ability of the antifungal compound to inhibit the growthof the transformed yeast cell. Such inhibition will occur if thesuspected Aspergillus nidulans MDR inhibitor blocks the ability of atrDto prevent the antifungal compound from acting on the yeast cell. Anillustrative example of such an assay is provided in Example 3.

In order to illustrate more fully the operation of this invention, thefollowing examples are provided, but are not to be construed as alimitation on the scope of the invention.

EXAMPLE 1 Source of the atrD-Encoding Genomic DNA and cDNA ofAspergillus nidulans

Genomic DNA encoding atrD, or the corresponding cDNA sequence (presentedin SEQ ID NO:1), may be from a natural sequence, a synthetic source or acombination of both (“semi-synthetic sequence”). The in vitro or in vivotranscription and translation of these sequences results in theproduction of atrD. Synthetic and semi-synthetic sequences encoding atrDmay be constructed by techniques well known in the art. See Brown et al.(1979) Methods in Enzymology, Academic Press, N.Y., 68:109-151.atrD-encoding DNA, or portions thereof, may be generated using aconventional DNA synthesizing apparatus such as the Applied BiosystemsModel 380A, 380B, 384 or 3848 DNA synthesizers (commercially availablefrom Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City,Calif. 94404). The polymerase chain reaction is especially useful ingenerating these DNA sequences. PCR primers are constructed whichinclude the translational start (ATG) and translational stop codon (TAG)of atrD. Restriction enzyme sites may be included on these PCR primersoutside of the atrD coding region to facilitate rapid cloning intoexpression vectors. Aspergillus nidulans genomic DNA is used as the PCRtemplate for synthesis of atrD including introns which is useful forexpression studies in closely related fungi. In contrast, cDNA is usedas the PCR template for synthesis of atrD devoid of introns which isuseful for expression in foreign hosts such as Saccharomyces cerevisiaeor bacterial hosts such as Escherichia coli.

EXAMPLE 2 Expression of the atrD Protein

Saccharomyces cerevisiae INVSc1 cells (Invitrogen Corp., San DiegoCalif. 92191) are transformed with the plasmid containing atrD by thetechnique described by J. D. Beggs, 1988, Nature 275:104-109). Thetransformed yeast cells are grown in a broth medium containingYNB/CSM-Ura/raf (YNB/CSM-Ura [Yeast Nitrogen Base (Difco Laboratories,Detroit, Mich.) supplemented with CSM-URA (Bio 101, Inc.)] supplementedwith 4% raffinose) at 28° C. in a shaker incubator until the culture issaturated. To induce expression of atrD, a portion of the culture isused to inoculate a flask containing YNB/CSM-Ura medium supplementedwith 2% galactose (YNB/CSM-Ura/gal) rather than raffinose as the solecarbon source. The inoculated flask is incubated at 28° C. for about 16hours.

EXAMPLE 3 Antifungal Potentiator Assay

Approximately 1×10⁶ cells of a Saccharomyces cerevisiae INVSc1 cultureexpressing atrD are delivered to each of several agar plates containingYNB/CSM-Ura/gal. The agar surface is allowed to dry in a biohazard hood.

An antifungal compound that the untransformed yeast cell is typicallysensitive to is dissolved in an appropriate solvent at a concentrationthat is biologically effective. Twenty μl of the solution is deliveredto an antibiotic susceptibility test disc (Difco Laboratories, Detroit,Mich.). After addition of the antifungal solution the disc is allowed toair dry in a biohazard hood. When dry, the disc is placed on the surfaceof the petri plates containing the transformed Saccharomyces cerevisiaeINVSc1 cells.

Compounds to be tested for the ability to inhibit atrD are dissolved indimethylsulfoxide (DMSO). The amount of compound added to the DMSOdepends on the solubility of the individual compound to be tested.Twenty μl of the suspensions containing a compound to be tested aredelivered to an antibiotic susceptibility test disc (Difco Laboratories,Detroit, Mich.). The disc is then placed on the surface of the driedpetri plates containing the transformed Saccharomyces cerevisiae INVSc1cells approximately 2 cm from the antifungal-containing disc. Petriplates containing the two discs are incubated at 28° C. for about 16-48hours.

Following this incubation period, the petri plates are examined forzones of growth inhibition around the discs. A zone of growth inhibitionnear the antifungal disc on the test plate indicates that the compoundbeing tested for MDR inhibition activity blocks the activity of atrD andallows the antifungal compound to inhibit the growth of the yeast hostcell. Such compounds are said to possess MDR inhibition activity. Littleor no zone of growth inhibition indicates that the test compound doesnot block MDR activity and, thus, atrD is allowed to act upon theantifungal compound to prevent its activity upon the host cell.

EXAMPLE 4 Screen For Novel Antifungal Compounds

A plasmid molecule is constructed which contains DNA sequenceinformation required for replication and genetic transformation in E.coli (e.g. ampicillin resistance). The plasmid also comprises DNAsequences encoding a marker for selection in fungal cells (e.g.hygromycin B phosphotransferase, phleomycin resistance, G418 resistance)under the control of an A. nidulans promoter. Additionally, the plasmidcontains an internal portion of the atrD gene (e.g. about 3000 basepairs which lack 500 base pairs at the N-terminal end, and about 500base pairs at the C-terminal end of the coding region specified by SEQID NO:1). The atrD gene fragment enables a single crossover genedisruption when transformed or otherwise introduced into A. nidulans.

Alternatively, a 5 kilobase pair to 6 kilobase pair region of A.nidulans genomic DNA containing the atrD gene is subcloned into theaforementioned plasmid. Then, a central portion of the atrD gene isremoved and replaced with a selectable marker, such as hyromycin Bphosphotransferase, for a double crossover gene replacement.

Gene disruption and gene replacement procedures for A. nidulans are wellknown in the art (See e.g. May et al, J. Cell Biol. 101, 712, 1985;Jones and Sealy-Lewis, Curr. Genet. 17, 81, 1990). Transformants arerecovered on an appropriate selection medium, for example, hygromycin(if hygromycin B gene is used in the construction of disruptioncassette). Gene replacement, or gene disruption, is verified by anysuitable method, for example, by Southern blot hybridization.

Gene disruption or gene replacement strains are rendered hypersensitiveto antifungal compounds, and are useful in screens for new antifungalcompounds in whole cell growth inhibition studies.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 4002 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO     (ix) FEATURE:           (A) NAME/KEY: CDS          (B) LOCATION: 1..4002    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:ATG TCC CCG CTA GAG ACA AAT CCC CTT TCG CC#A GAG ACT GCT ATG CGC       48Met Ser Pro Leu Glu Thr Asn Pro Leu Ser Pr #o Glu Thr Ala Met Arg  1               5  #                 10  #                 15GAA CCT GCT GAG ACT TCA ACG ACG GAG GAG CA#A GCT TCT ACA CCA CAC       96Glu Pro Ala Glu Thr Ser Thr Thr Glu Glu Gl #n Ala Ser Thr Pro His             20      #             25      #             30GCT GCG GAC GAG AAG AAA ATC CTC AGC GAC CT#C TCG GCT CCA TCT AGT      144Ala Ala Asp Glu Lys Lys Ile Leu Ser Asp Le #u Ser Ala Pro Ser Ser         35          #         40          #         45ACT ACA GCA ACC CCC GCA GAC AAG GAG CAC CG#T CCT AAA TCG TCG TCC      192Thr Thr Ala Thr Pro Ala Asp Lys Glu His Ar #g Pro Lys Ser Ser Ser     50              #     55              #     60AGC AAT AAT GCG GTC TCG GTC AAC GAA GTC GA#T GCG CTT ATT GCG CAC      240Ser Asn Asn Ala Val Ser Val Asn Glu Val As #p Ala Leu Ile Ala His 65                  # 70                  # 75                  # 80CTG CCA GAA GAC GAG AGG CAG GTC TTG AAG AC#G CAG CTG GAG GAG ATC      288Leu Pro Glu Asp Glu Arg Gln Val Leu Lys Th #r Gln Leu Glu Glu Ile                 85  #                 90  #                 95AAA GTA AAC ATC TCC TTC TTC GGT CTC TGG CG#G TAT GCA ACA AAG ATG      336Lys Val Asn Ile Ser Phe Phe Gly Leu Trp Ar #g Tyr Ala Thr Lys Met            100       #           105       #           110GAT ATA CTT ATC ATG GTA ATC AGT ACA ATC TG#T GCC ATT GCT GCC GCG      384Asp Ile Leu Ile Met Val Ile Ser Thr Ile Cy #s Ala Ile Ala Ala Ala        115           #       120           #       125TCG ACT TTC CAG AGG ATA ATG TTA TAT CAA AT#C TCG TAC GAC GAG TTC      432Ser Thr Phe Gln Arg Ile Met Leu Tyr Gln Il #e Ser Tyr Asp Glu Phe    130               #   135               #   140TAT GAT GAA TTG ACC AAG AAC GTA CTG TAC TT#C GTA TAC CTC GGT ATC      480Tyr Asp Glu Leu Thr Lys Asn Val Leu Tyr Ph #e Val Tyr Leu Gly Ile145                 1 #50                 1 #55                 1 #60GGC GAG TTT GTC ACT GTC TAT GTT AGT ACT GT#T GGC TTC ATC TAT ACC      528Gly Glu Phe Val Thr Val Tyr Val Ser Thr Va #l Gly Phe Ile Tyr Thr                165   #               170   #               175GGA GAA CAC GCC ACG CAG AAG ATC CGC GAG TA#T TAC CTT GAG TCT ATC      576Gly Glu His Ala Thr Gln Lys Ile Arg Glu Ty #r Tyr Leu Glu Ser Ile            180       #           185       #           190CTG CGC CAG AAC ATT GGC TAT TTT GAT AAA CT#C GGT GCC GGG GAA GTG      624Leu Arg Gln Asn Ile Gly Tyr Phe Asp Lys Le #u Gly Ala Gly Glu Val        195           #       200           #       205ACC ACC CGT ATA ACA GCC GAT ACA AAC CTT AT#C CAG GAT GGC ATT TCG      672Thr Thr Arg Ile Thr Ala Asp Thr Asn Leu Il #e Gln Asp Gly Ile Ser    210               #   215               #   220GAG AAG GTC GGT CTC ACT TTG ACT GCC CTG GC#G ACA TTC GTG ACA GCA      720Glu Lys Val Gly Leu Thr Leu Thr Ala Leu Al #a Thr Phe Val Thr Ala225                 2 #30                 2 #35                 2 #40TTC ATT ATC GCC TAC GTC AAA TAC TGG AAG TT#G GCT CTA ATT TGC AGC      768Phe Ile Ile Ala Tyr Val Lys Tyr Trp Lys Le #u Ala Leu Ile Cys Ser                245   #               250   #               255TCA ACA ATT GTG GCC CTC GTT CTC ACC ATG GG#C GGT GGT TCT CAG TTT      816Ser Thr Ile Val Ala Leu Val Leu Thr Met Gl #y Gly Gly Ser Gln Phe            260       #           265       #           270ATC ATC AAG TAC AGC AAA AAG TCG CTT GAC AG#C TAC GGT GCA GGC GGC      864Ile Ile Lys Tyr Ser Lys Lys Ser Leu Asp Se #r Tyr Gly Ala Gly Gly        275           #       280           #       285ACT GTT GCG GAA GAG GTC ATC AGC TCC ATC AG#A AAT GCC ACA GCG TTT      912Thr Val Ala Glu Glu Val Ile Ser Ser Ile Ar #g Asn Ala Thr Ala Phe    290               #   295               #   300GGC ACC CAA GAC AAG CTT GCG AAG CAG TAT GA#G GTC CAC TTA GAC GAA      960Gly Thr Gln Asp Lys Leu Ala Lys Gln Tyr Gl #u Val His Leu Asp Glu305                 3 #10                 3 #15                 3 #20GCT GAG AAA TGG GGA ACA AAG AAC CAG ATT GT#C ATG GGT TTC ATG ATT     1008Ala Glu Lys Trp Gly Thr Lys Asn Gln Ile Va #l Met Gly Phe Met Ile                325   #               330   #               335GGC GCC ATG TTT GGC CTT ATG TAC TCG AAC TA#C GGT CTT GGC TTC TGG     1056Gly Ala Met Phe Gly Leu Met Tyr Ser Asn Ty #r Gly Leu Gly Phe Trp            340       #           345       #           350ATG GGT TCT CGT TTC CTG GTA GAT GGT GCA GT#C GAT GTG GGT GAT ATT     1104Met Gly Ser Arg Phe Leu Val Asp Gly Ala Va #l Asp Val Gly Asp Ile        355           #       360           #       365CTC ACA GTT CTC ATG GCC ATC TTG ATC GGA TC#G TTC TCC TTG GGG AAC     1152Leu Thr Val Leu Met Ala Ile Leu Ile Gly Se #r Phe Ser Leu Gly Asn    370               #   375               #   380GTT AGT CCA AAT GCT CAA GCA TTT ACA AAC GC#T GTG GCC GCG GCC GCA     1200Val Ser Pro Asn Ala Gln Ala Phe Thr Asn Al #a Val Ala Ala Ala Ala385                 3 #90                 3 #95                 4 #00AAG ATA TTT GGA ACG ATC GAT CGC CAG TCC CC#A TTA GAT CCA TAT TCG     1248Lys Ile Phe Gly Thr Ile Asp Arg Gln Ser Pr #o Leu Asp Pro Tyr Ser                405   #               410   #               415AAC GAA GGG AAG ACG CTC GAC CAT TTT GAG GG#C CAC ATT GAG TTA CGC     1296Asn Glu Gly Lys Thr Leu Asp His Phe Glu Gl #y His Ile Glu Leu Arg            420       #           425       #           430AAT GTC AAG CAT ATT TAC CCA TCT AGA CCC GA#G GTC ACC GTC ATG GAG     1344Asn Val Lys His Ile Tyr Pro Ser Arg Pro Gl #u Val Thr Val Met Glu        435           #       440           #       445GAT GTT TCT CTG TCA ATG CCC GCT GGA AAA AC#A ACC GCT TTA GTC GGC     1392Asp Val Ser Leu Ser Met Pro Ala Gly Lys Th #r Thr Ala Leu Val Gly    450               #   455               #   460CCC TCT GGC TCT GGA AAA AGT ACG GTG GTC GG#C TTG GTT GAG CGA TTC     1440Pro Ser Gly Ser Gly Lys Ser Thr Val Val Gl #y Leu Val Glu Arg Phe465                 4 #70                 4 #75                 4 #80TAC ATG CCT GTT CGC GGT ACG GTT TTG CTG GA#T GGC CAT GAC ATC AAG     1488Tyr Met Pro Val Arg Gly Thr Val Leu Leu As #p Gly His Asp Ile Lys                485   #               490   #               495GAC CTC AAT CTC CGC TGG CTT CGC CAA CAG AT#C TCT TTG GTT AGC CAG     1536Asp Leu Asn Leu Arg Trp Leu Arg Gln Gln Il #e Ser Leu Val Ser Gln            500       #           505       #           510GAG CCT GTT CTT TTT GGC ACG ACG ATT TAT AA#G AAT ATT AGG CAC GGT     1584Glu Pro Val Leu Phe Gly Thr Thr Ile Tyr Ly #s Asn Ile Arg His Gly        515           #       520           #       525CTC ATC GGC ACA AAG TAC GAG AAT GAA TCC GA#G GAT AAG GTC CGG GAA     1632Leu Ile Gly Thr Lys Tyr Glu Asn Glu Ser Gl #u Asp Lys Val Arg Glu    530               #   535               #   540CTC ATC GAG AAC GCG GCA AAA ATG GCG AAT GC#T CAT GAC TTT ATT ACT     1680Leu Ile Glu Asn Ala Ala Lys Met Ala Asn Al #a His Asp Phe Ile Thr545                 5 #50                 5 #55                 5 #60GCC TTG CCT GAA GGT TAT GAG ACC AAT GTT GG#G CAG CGT GGC TTT CTC     1728Ala Leu Pro Glu Gly Tyr Glu Thr Asn Val Gl #y Gln Arg Gly Phe Leu                565   #               570   #               575CTT TCA GGT GGC CAG AAA CAG CGC ATT GCA AT#C GCC CGT GCC GTT GTT     1776Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Il #e Ala Arg Ala Val Val            580       #           585       #           590AGT GAC CCA AAA ATC CTG CTC CTG GAT GAA GC#T ACT TCG GCC TTG GAC     1824Ser Asp Pro Lys Ile Leu Leu Leu Asp Glu Al #a Thr Ser Ala Leu Asp        595           #       600           #       605ACA AAA TCC GAA GGC GTG GTT CAA GCA GCT TT#G GAG AGG GCA GCT GAA     1872Thr Lys Ser Glu Gly Val Val Gln Ala Ala Le #u Glu Arg Ala Ala Glu    610               #   615               #   620GGC CGA ACT ACT ATT GTG ATC GCT CAT CGC CT#T TCC ACG ATC AAA ACG     1920Gly Arg Thr Thr Ile Val Ile Ala His Arg Le #u Ser Thr Ile Lys Thr625                 6 #30                 6 #35                 6 #40GCG CAC AAC ATT GTG GTT CTG GTC AAT GGC AA#A ATT GCT GAA CAA GGA     1968Ala His Asn Ile Val Val Leu Val Asn Gly Ly #s Ile Ala Glu Gln Gly                645   #               650   #               655ACT CAC GAT GAA TTG GTT GAC CGC GGA GGC GC#T TAT CGC AAA CTT GTG     2016Thr His Asp Glu Leu Val Asp Arg Gly Gly Al #a Tyr Arg Lys Leu Val            660       #           665       #           670GAG GCT CAA CGT ATC AAT GAA CAG AAG GAA GC#T GAC GCC TTG GAG GAC     2064Glu Ala Gln Arg Ile Asn Glu Gln Lys Glu Al #a Asp Ala Leu Glu Asp        675           #       680           #       685GCC GAC GCT GAG GAT CTC ACG AAT GCA GAT AT#T GCC AAA ATC AAA ACT     2112Ala Asp Ala Glu Asp Leu Thr Asn Ala Asp Il #e Ala Lys Ile Lys Thr    690               #   695               #   700GCG TCA AGC GCA TCA TCC GAT CTC GAC GGA AA#A CCC ACA ACC ATT GAC     2160Ala Ser Ser Ala Ser Ser Asp Leu Asp Gly Ly #s Pro Thr Thr Ile Asp705                 7 #10                 7 #15                 7 #20CGC ACG GGC ACC CAC AAG TCT GTT TCC AGC GC#G ATT CTT TCT AAA AGA     2208Arg Thr Gly Thr His Lys Ser Val Ser Ser Al #a Ile Leu Ser Lys Arg                725   #               730   #               735CCC CCC GAA ACA ACT CCG AAA TAC TCA TTA TG#G ACG CTG CTC AAA TTT     2256Pro Pro Glu Thr Thr Pro Lys Tyr Ser Leu Tr #p Thr Leu Leu Lys Phe            740       #           745       #           750GTT GCT TCC TTC AAC CGC CCT GAA ATC CCG TA#C ATG CTC ATC GGT CTT     2304Val Ala Ser Phe Asn Arg Pro Glu Ile Pro Ty #r Met Leu Ile Gly Leu        755           #       760           #       765GTC TTC TCA GTG TTA GCT GGT GGT GGC CAA CC#C ACG CAA GCA GTG CTA     2352Val Phe Ser Val Leu Ala Gly Gly Gly Gln Pr #o Thr Gln Ala Val Leu    770               #   775               #   780TAT GCT AAA GCC ATC AGC ACA CTC TCG CTC CC#A GAA TCA CAA TAT AGC     2400Tyr Ala Lys Ala Ile Ser Thr Leu Ser Leu Pr #o Glu Ser Gln Tyr Ser785                 7 #90                 7 #95                 8 #00AAG CTT CGA CAT GAT GCG GAT TTC TGG TCA TT#G ATG TTC TTC GTG GTT     2448Lys Leu Arg His Asp Ala Asp Phe Trp Ser Le #u Met Phe Phe Val Val                805   #               810   #               815GGT ATC ATT CAG TTT ATC ACG CAG TCA ACC AA#T GGT GCT GCA TTT GCC     2496Gly Ile Ile Gln Phe Ile Thr Gln Ser Thr As #n Gly Ala Ala Phe Ala            820       #           825       #           830GTA TGC TCC GAG AGA CTT ATT CGT CGC GCG AG#A AGC ACT GCC TTT CGG     2544Val Cys Ser Glu Arg Leu Ile Arg Arg Ala Ar #g Ser Thr Ala Phe Arg        835           #       840           #       845ACG ATA CTC CGT CAA GAC ATT GCT TTC TTT GA#C AAG GAA GAG AAT AGC     2592Thr Ile Leu Arg Gln Asp Ile Ala Phe Phe As #p Lys Glu Glu Asn Ser    850               #   855               #   860ACC GGC GCT CTG ACC TCT TTC CTG TCC ACC GA#G ACG AAG CAT CTC TCC     2640Thr Gly Ala Leu Thr Ser Phe Leu Ser Thr Gl #u Thr Lys His Leu Ser865                 8 #70                 8 #75                 8 #80GGT GTT AGC GGT GTG ACT CTA GGC ACG ATC TT#G ATG ACC TCC ACG ACC     2688Gly Val Ser Gly Val Thr Leu Gly Thr Ile Le #u Met Thr Ser Thr Thr                885   #               890   #               895CTA GGA GCG GCT ATC ATT ATT GCC CTG GCG AT#T GGG TGG AAA TTG GCC     2736Leu Gly Ala Ala Ile Ile Ile Ala Leu Ala Il #e Gly Trp Lys Leu Ala            900       #           905       #           910TTA GTT TGT ATC TCG GTT GTG CCG GTT CTC CT#G GCA TGC GGT TTC TAC     2784Leu Val Cys Ile Ser Val Val Pro Val Leu Le #u Ala Cys Gly Phe Tyr        915           #       920           #       925CGA TTC TAT ATG CTA GCC CAG TTT CAA TCA CG#C TCC AAG CTT GCT TAT     2832Arg Phe Tyr Met Leu Ala Gln Phe Gln Ser Ar #g Ser Lys Leu Ala Tyr    930               #   935               #   940GAG GGA TCT GCA AAC TTT GCT TGC GAG GCT AC#A TCG TCT ATC CGC ACA     2880Glu Gly Ser Ala Asn Phe Ala Cys Glu Ala Th #r Ser Ser Ile Arg Thr945                 9 #50                 9 #55                 9 #60GTT GCG TCA TTA ACC CGG GAA AGG GAT GTC TG#G GAG ATT TAC CAT GCC     2928Val Ala Ser Leu Thr Arg Glu Arg Asp Val Tr #p Glu Ile Tyr His Ala                965   #               970   #               975CAG CTT GAC GCA CAA GGC AGG ACC AGT CTA AT#C TCT GTC TTG AGG TCA     2976Gln Leu Asp Ala Gln Gly Arg Thr Ser Leu Il #e Ser Val Leu Arg Ser            980       #           985       #           990TCC CTG TTA TAT GCG TCG TCG CAG GCA CTT GT#T TTC TTC TGC GTT GCG     3024Ser Leu Leu Tyr Ala Ser Ser Gln Ala Leu Va #l Phe Phe Cys Val Ala        995           #       1000           #      1005CTC GGG TTT TGG TAC GGA GGG ACA CTT CTT GG#T CAC CAC GAG TAT GAC     3072Leu Gly Phe Trp Tyr Gly Gly Thr Leu Leu Gl #y His His Glu Tyr Asp    1010              #   1015               #  1020ATT TTC CGC TTC TTT GTT TGT TTC TCC GAG AT#T CTC TTT GGT GCT CAA     3120Ile Phe Arg Phe Phe Val Cys Phe Ser Glu Il #e Leu Phe Gly Ala Gln1025                1030 #                1035  #               1040TCC GCG GGC ACC GTC TTT TCC TTT GCA CCA GA#C ATG GGC AAG GCG AAG     3168Ser Ala Gly Thr Val Phe Ser Phe Ala Pro As #p Met Gly Lys Ala Lys                1045  #               1050   #              1055AAT GCG GCC GCC GAA TTC CGA CGA CTG TTC GA#C CGA AAG CCA CAA ATT     3216Asn Ala Ala Ala Glu Phe Arg Arg Leu Phe As #p Arg Lys Pro Gln Ile            1060      #           1065       #          1070GAT AAC TGG TCT GAA GAG GGC GAG AAG CTC GA#A ACG GTG GAA GGT GAA     3264Asp Asn Trp Ser Glu Glu Gly Glu Lys Leu Gl #u Thr Val Glu Gly Glu        1075          #       1080           #      1085ATC GAA TTT AGG AAC GTG CAC TTC AGA TAC CC#G ACC CGC CCA GAA CAG     3312Ile Glu Phe Arg Asn Val His Phe Arg Tyr Pr #o Thr Arg Pro Glu Gln    1090              #   1095               #  1100CCT GTC CTG CGC GGC TTG GAC CTG ACC GTG AA#G CCT GGA CAA TAT GTT     3360Pro Val Leu Arg Gly Leu Asp Leu Thr Val Ly #s Pro Gly Gln Tyr Val1105                1110 #                1115  #               1120GCG CTT GTC GGA CCC AGC GGT TGT GGC AAG AG#T ACC ACC ATT GCA TTG     3408Ala Leu Val Gly Pro Ser Gly Cys Gly Lys Se #r Thr Thr Ile Ala Leu                1125  #               1130   #              1135CTT GAG CGC TTT TAC GAT GCG ATT GCC GGG TC#C ATC CTT GTT GAT GGG     3456Leu Glu Arg Phe Tyr Asp Ala Ile Ala Gly Se #r Ile Leu Val Asp Gly            1140      #           1145       #          1150AAG GAC ATA AGT AAA CTA AAT ATC AAC TCC TA#C CGC AGC TTT CTG TCA     3504Lys Asp Ile Ser Lys Leu Asn Ile Asn Ser Ty #r Arg Ser Phe Leu Ser        1155          #       1160           #      1165CTG GTC AGC CAG GAG CCG ACA CTG TAC CAG GG#C ACC ATC AAG GAA AAC     3552Leu Val Ser Gln Glu Pro Thr Leu Tyr Gln Gl #y Thr Ile Lys Glu Asn    1170              #   1175               #  1180ATC TTA CTT GGT ATT GTC GAA GAT GAC GTA CC#G GAA GAA TTC TTG ATT     3600Ile Leu Leu Gly Ile Val Glu Asp Asp Val Pr #o Glu Glu Phe Leu Ile1185                1190 #                1195  #               1200AAG GCT TGC AAG GAC GCT AAT ATC TAC GAC TT#C ATC ATG TCG CTC CCG     3648Lys Ala Cys Lys Asp Ala Asn Ile Tyr Asp Ph #e Ile Met Ser Leu Pro                1205  #               1210   #              1215GAG GGC TTT AAT ACA GTT GTT GGC AGC AAG GG#A GGC ATG TTG TCT GGC     3696Glu Gly Phe Asn Thr Val Val Gly Ser Lys Gl #y Gly Met Leu Ser Gly            1220      #           1225       #          1230GGC CAA AAG CAA CGT GTG GCC ATT GCC CGA GC#C CTT CTT CGG GAT CCC     3744Gly Gln Lys Gln Arg Val Ala Ile Ala Arg Al #a Leu Leu Arg Asp Pro        1235          #       1240           #      1245AAA ATC CTT CTT CTC GAT GAA GCG ACG TCA GC#C CTC GAC TCC GAG TCA     3792Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Al #a Leu Asp Ser Glu Ser    1250              #   1255               #  1260GAA AAG GTC GTC CAG GCG GCT TTG GAT GCC GC#T GCC CGA GGC CGA ACC     3840Glu Lys Val Val Gln Ala Ala Leu Asp Ala Al #a Ala Arg Gly Arg Thr1265                1270 #                1275  #               1280ACA ATC GCC GTT GCA CAC CGA CTC AGC ACG AT#T CAA AAG GCG GAC GTT     3888Thr Ile Ala Val Ala His Arg Leu Ser Thr Il #e Gln Lys Ala Asp Val                1285  #               1290   #              1295ATC TAT GTT TTC GAC CAA GGC AAG ATC GTC GA#A AGC GGA ACG CAC AGC     3936Ile Tyr Val Phe Asp Gln Gly Lys Ile Val Gl #u Ser Gly Thr His Ser            1300      #           1305       #          1310GAA CTG GTC CAG AAA AAG GGC CGG TAC TAC GA#G CTG GTC AAC TTG CAG     3984Glu Leu Val Gln Lys Lys Gly Arg Tyr Tyr Gl #u Leu Val Asn Leu Gln        1315          #       1320           #      1325AGC TTG GGC AAG GGC CAT          #                   #                  #4002 Ser Leu Gly Lys Gly His     1330 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 1334 amino #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:Met Ser Pro Leu Glu Thr Asn Pro Leu Ser Pr #o Glu Thr Ala Met Arg  1               5  #                 10  #                 15Glu Pro Ala Glu Thr Ser Thr Thr Glu Glu Gl #n Ala Ser Thr Pro His             20      #             25      #             30Ala Ala Asp Glu Lys Lys Ile Leu Ser Asp Le #u Ser Ala Pro Ser Ser         35          #         40          #         45Thr Thr Ala Thr Pro Ala Asp Lys Glu His Ar #g Pro Lys Ser Ser Ser     50              #     55              #     60Ser Asn Asn Ala Val Ser Val Asn Glu Val As #p Ala Leu Ile Ala His 65                  # 70                  # 75                  # 80Leu Pro Glu Asp Glu Arg Gln Val Leu Lys Th #r Gln Leu Glu Glu Ile                 85  #                 90  #                 95Lys Val Asn Ile Ser Phe Phe Gly Leu Trp Ar #g Tyr Ala Thr Lys Met            100       #           105       #           110Asp Ile Leu Ile Met Val Ile Ser Thr Ile Cy #s Ala Ile Ala Ala Ala        115           #       120           #       125Ser Thr Phe Gln Arg Ile Met Leu Tyr Gln Il #e Ser Tyr Asp Glu Phe    130               #   135               #   140Tyr Asp Glu Leu Thr Lys Asn Val Leu Tyr Ph #e Val Tyr Leu Gly Ile145                 1 #50                 1 #55                 1 #60Gly Glu Phe Val Thr Val Tyr Val Ser Thr Va #l Gly Phe Ile Tyr Thr                165   #               170   #               175Gly Glu His Ala Thr Gln Lys Ile Arg Glu Ty #r Tyr Leu Glu Ser Ile            180       #           185       #           190Leu Arg Gln Asn Ile Gly Tyr Phe Asp Lys Le #u Gly Ala Gly Glu Val        195           #       200           #       205Thr Thr Arg Ile Thr Ala Asp Thr Asn Leu Il #e Gln Asp Gly Ile Ser    210               #   215               #   220Glu Lys Val Gly Leu Thr Leu Thr Ala Leu Al #a Thr Phe Val Thr Ala225                 2 #30                 2 #35                 2 #40Phe Ile Ile Ala Tyr Val Lys Tyr Trp Lys Le #u Ala Leu Ile Cys Ser                245   #               250   #               255Ser Thr Ile Val Ala Leu Val Leu Thr Met Gl #y Gly Gly Ser Gln Phe            260       #           265       #           270Ile Ile Lys Tyr Ser Lys Lys Ser Leu Asp Se #r Tyr Gly Ala Gly Gly        275           #       280           #       285Thr Val Ala Glu Glu Val Ile Ser Ser Ile Ar #g Asn Ala Thr Ala Phe    290               #   295               #   300Gly Thr Gln Asp Lys Leu Ala Lys Gln Tyr Gl #u Val His Leu Asp Glu305                 3 #10                 3 #15                 3 #20Ala Glu Lys Trp Gly Thr Lys Asn Gln Ile Va #l Met Gly Phe Met Ile                325   #               330   #               335Gly Ala Met Phe Gly Leu Met Tyr Ser Asn Ty #r Gly Leu Gly Phe Trp            340       #           345       #           350Met Gly Ser Arg Phe Leu Val Asp Gly Ala Va #l Asp Val Gly Asp Ile        355           #       360           #       365Leu Thr Val Leu Met Ala Ile Leu Ile Gly Se #r Phe Ser Leu Gly Asn    370               #   375               #   380Val Ser Pro Asn Ala Gln Ala Phe Thr Asn Al #a Val Ala Ala Ala Ala385                 3 #90                 3 #95                 4 #00Lys Ile Phe Gly Thr Ile Asp Arg Gln Ser Pr #o Leu Asp Pro Tyr Ser                405   #               410   #               415Asn Glu Gly Lys Thr Leu Asp His Phe Glu Gl #y His Ile Glu Leu Arg            420       #           425       #           430Asn Val Lys His Ile Tyr Pro Ser Arg Pro Gl #u Val Thr Val Met Glu        435           #       440           #       445Asp Val Ser Leu Ser Met Pro Ala Gly Lys Th #r Thr Ala Leu Val Gly    450               #   455               #   460Pro Ser Gly Ser Gly Lys Ser Thr Val Val Gl #y Leu Val Glu Arg Phe465                 4 #70                 4 #75                 4 #80Tyr Met Pro Val Arg Gly Thr Val Leu Leu As #p Gly His Asp Ile Lys                485   #               490   #               495Asp Leu Asn Leu Arg Trp Leu Arg Gln Gln Il #e Ser Leu Val Ser Gln            500       #           505       #           510Glu Pro Val Leu Phe Gly Thr Thr Ile Tyr Ly #s Asn Ile Arg His Gly        515           #       520           #       525Leu Ile Gly Thr Lys Tyr Glu Asn Glu Ser Gl #u Asp Lys Val Arg Glu    530               #   535               #   540Leu Ile Glu Asn Ala Ala Lys Met Ala Asn Al #a His Asp Phe Ile Thr545                 5 #50                 5 #55                 5 #60Ala Leu Pro Glu Gly Tyr Glu Thr Asn Val Gl #y Gln Arg Gly Phe Leu                565   #               570   #               575Leu Ser Gly Gly Gln Lys Gln Arg Ile Ala Il #e Ala Arg Ala Val Val            580       #           585       #           590Ser Asp Pro Lys Ile Leu Leu Leu Asp Glu Al #a Thr Ser Ala Leu Asp        595           #       600           #       605Thr Lys Ser Glu Gly Val Val Gln Ala Ala Le #u Glu Arg Ala Ala Glu    610               #   615               #   620Gly Arg Thr Thr Ile Val Ile Ala His Arg Le #u Ser Thr Ile Lys Thr625                 6 #30                 6 #35                 6 #40Ala His Asn Ile Val Val Leu Val Asn Gly Ly #s Ile Ala Glu Gln Gly                645   #               650   #               655Thr His Asp Glu Leu Val Asp Arg Gly Gly Al #a Tyr Arg Lys Leu Val            660       #           665       #           670Glu Ala Gln Arg Ile Asn Glu Gln Lys Glu Al #a Asp Ala Leu Glu Asp        675           #       680           #       685Ala Asp Ala Glu Asp Leu Thr Asn Ala Asp Il #e Ala Lys Ile Lys Thr    690               #   695               #   700Ala Ser Ser Ala Ser Ser Asp Leu Asp Gly Ly #s Pro Thr Thr Ile Asp705                 7 #10                 7 #15                 7 #20Arg Thr Gly Thr His Lys Ser Val Ser Ser Al #a Ile Leu Ser Lys Arg                725   #               730   #               735Pro Pro Glu Thr Thr Pro Lys Tyr Ser Leu Tr #p Thr Leu Leu Lys Phe            740       #           745       #           750Val Ala Ser Phe Asn Arg Pro Glu Ile Pro Ty #r Met Leu Ile Gly Leu        755           #       760           #       765Val Phe Ser Val Leu Ala Gly Gly Gly Gln Pr #o Thr Gln Ala Val Leu    770               #   775               #   780Tyr Ala Lys Ala Ile Ser Thr Leu Ser Leu Pr #o Glu Ser Gln Tyr Ser785                 7 #90                 7 #95                 8 #00Lys Leu Arg His Asp Ala Asp Phe Trp Ser Le #u Met Phe Phe Val Val                805   #               810   #               815Gly Ile Ile Gln Phe Ile Thr Gln Ser Thr As #n Gly Ala Ala Phe Ala            820       #           825       #           830Val Cys Ser Glu Arg Leu Ile Arg Arg Ala Ar #g Ser Thr Ala Phe Arg        835           #       840           #       845Thr Ile Leu Arg Gln Asp Ile Ala Phe Phe As #p Lys Glu Glu Asn Ser    850               #   855               #   860Thr Gly Ala Leu Thr Ser Phe Leu Ser Thr Gl #u Thr Lys His Leu Ser865                 8 #70                 8 #75                 8 #80Gly Val Ser Gly Val Thr Leu Gly Thr Ile Le #u Met Thr Ser Thr Thr                885   #               890   #               895Leu Gly Ala Ala Ile Ile Ile Ala Leu Ala Il #e Gly Trp Lys Leu Ala            900       #           905       #           910Leu Val Cys Ile Ser Val Val Pro Val Leu Le #u Ala Cys Gly Phe Tyr        915           #       920           #       925Arg Phe Tyr Met Leu Ala Gln Phe Gln Ser Ar #g Ser Lys Leu Ala Tyr    930               #   935               #   940Glu Gly Ser Ala Asn Phe Ala Cys Glu Ala Th #r Ser Ser Ile Arg Thr945                 9 #50                 9 #55                 9 #60Val Ala Ser Leu Thr Arg Glu Arg Asp Val Tr #p Glu Ile Tyr His Ala                965   #               970   #               975Gln Leu Asp Ala Gln Gly Arg Thr Ser Leu Il #e Ser Val Leu Arg Ser            980       #           985       #           990Ser Leu Leu Tyr Ala Ser Ser Gln Ala Leu Va #l Phe Phe Cys Val Ala        995           #       1000           #      1005Leu Gly Phe Trp Tyr Gly Gly Thr Leu Leu Gl #y His His Glu Tyr Asp    1010              #   1015               #  1020Ile Phe Arg Phe Phe Val Cys Phe Ser Glu Il #e Leu Phe Gly Ala Gln1025                1030 #                1035  #               1040Ser Ala Gly Thr Val Phe Ser Phe Ala Pro As #p Met Gly Lys Ala Lys                1045  #               1050   #              1055Asn Ala Ala Ala Glu Phe Arg Arg Leu Phe As #p Arg Lys Pro Gln Ile            1060      #           1065       #          1070Asp Asn Trp Ser Glu Glu Gly Glu Lys Leu Gl #u Thr Val Glu Gly Glu        1075          #       1080           #      1085Ile Glu Phe Arg Asn Val His Phe Arg Tyr Pr #o Thr Arg Pro Glu Gln    1090              #   1095               #  1100Pro Val Leu Arg Gly Leu Asp Leu Thr Val Ly #s Pro Gly Gln Tyr Val1105                1110 #                1115  #               1120Ala Leu Val Gly Pro Ser Gly Cys Gly Lys Se #r Thr Thr Ile Ala Leu                1125  #               1130   #              1135Leu Glu Arg Phe Tyr Asp Ala Ile Ala Gly Se #r Ile Leu Val Asp Gly            1140      #           1145       #          1150Lys Asp Ile Ser Lys Leu Asn Ile Asn Ser Ty #r Arg Ser Phe Leu Ser        1155          #       1160           #      1165Leu Val Ser Gln Glu Pro Thr Leu Tyr Gln Gl #y Thr Ile Lys Glu Asn    1170              #   1175               #  1180Ile Leu Leu Gly Ile Val Glu Asp Asp Val Pr #o Glu Glu Phe Leu Ile1185                1190 #                1195  #               1200Lys Ala Cys Lys Asp Ala Asn Ile Tyr Asp Ph #e Ile Met Ser Leu Pro                1205  #               1210   #              1215Glu Gly Phe Asn Thr Val Val Gly Ser Lys Gl #y Gly Met Leu Ser Gly            1220      #           1225       #          1230Gly Gln Lys Gln Arg Val Ala Ile Ala Arg Al #a Leu Leu Arg Asp Pro        1235          #       1240           #      1245Lys Ile Leu Leu Leu Asp Glu Ala Thr Ser Al #a Leu Asp Ser Glu Ser    1250              #   1255               #  1260Glu Lys Val Val Gln Ala Ala Leu Asp Ala Al #a Ala Arg Gly Arg Thr1265                1270 #                1275  #               1280Thr Ile Ala Val Ala His Arg Leu Ser Thr Il #e Gln Lys Ala Asp Val                1285  #               1290   #              1295Ile Tyr Val Phe Asp Gln Gly Lys Ile Val Gl #u Ser Gly Thr His Ser            1300      #           1305       #          1310Glu Leu Val Gln Lys Lys Gly Arg Tyr Tyr Gl #u Leu Val Asn Leu Gln        1315          #       1320           #      1325Ser Leu Gly Lys Gly His     1330 (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 4002 base #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: mRNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:AUGUCCCCGC UAGAGACAAA UCCCCUUUCG CCAGAGACUG CUAUGCGCGA AC#CUGCUGAG     60ACUUCAACGA CGGAGGAGCA AGCUUCUACA CCACACGCUG CGGACGAGAA GA#AAAUCCUC    120AGCGACCUCU CGGCUCCAUC UAGUACUACA GCAACCCCCG CAGACAAGGA GC#ACCGUCCU    180AAAUCGUCGU CCAGCAAUAA UGCGGUCUCG GUCAACGAAG UCGAUGCGCU UA#UUGCGCAC    240CUGCCAGAAG ACGAGAGGCA GGUCUUGAAG ACGCAGCUGG AGGAGAUCAA AG#UAAACAUC    300UCCUUCUUCG GUCUCUGGCG GUAUGCAACA AAGAUGGAUA UACUUAUCAU GG#UAAUCAGU    360ACAAUCUGUG CCAUUGCUGC CGCGUCGACU UUCCAGAGGA UAAUGUUAUA UC#AAAUCUCG    420UACGACGAGU UCUAUGAUGA AUUGACCAAG AACGUACUGU ACUUCGUAUA CC#UCGGUAUC    480GGCGAGUUUG UCACUGUCUA UGUUAGUACU GUUGGCUUCA UCUAUACCGG AG#AACACGCC    540ACGCAGAAGA UCCGCGAGUA UUACCUUGAG UCUAUCCUGC GCCAGAACAU UG#GCUAUUUU    600GAUAAACUCG GUGCCGGGGA AGUGACCACC CGUAUAACAG CCGAUACAAA CC#UUAUCCAG    660GAUGGCAUUU CGGAGAAGGU CGGUCUCACU UUGACUGCCC UGGCGACAUU CG#UGACAGCA    720UUCAUUAUCG CCUACGUCAA AUACUGGAAG UUGGCUCUAA UUUGCAGCUC AA#CAAUUGUG    780GCCCUCGUUC UCACCAUGGG CGGUGGUUCU CAGUUUAUCA UCAAGUACAG CA#AAAAGUCG    840CUUGACAGCU ACGGUGCAGG CGGCACUGUU GCGGAAGAGG UCAUCAGCUC CA#UCAGAAAU    900GCCACAGCGU UUGGCACCCA AGACAAGCUU GCGAAGCAGU AUGAGGUCCA CU#UAGACGAA    960GCUGAGAAAU GGGGAACAAA GAACCAGAUU GUCAUGGGUU UCAUGAUUGG CG#CCAUGUUU   1020GGCCUUAUGU ACUCGAACUA CGGUCUUGGC UUCUGGAUGG GUUCUCGUUU CC#UGGUAGAU   1080GGUGCAGUCG AUGUGGGUGA UAUUCUCACA GUUCUCAUGG CCAUCUUGAU CG#GAUCGUUC   1140UCCUUGGGGA ACGUUAGUCC AAAUGCUCAA GCAUUUACAA ACGCUGUGGC CG#CGGCCGCA   1200AAGAUAUUUG GAACGAUCGA UCGCCAGUCC CCAUUAGAUC CAUAUUCGAA CG#AAGGGAAG   1260ACGCUCGACC AUUUUGAGGG CCACAUUGAG UUACGCAAUG UCAAGCAUAU UU#ACCCAUCU   1320AGACCCGAGG UCACCGUCAU GGAGGAUGUU UCUCUGUCAA UGCCCGCUGG AA#AAACAACC   1380GCUUUAGUCG GCCCCUCUGG CUCUGGAAAA AGUACGGUGG UCGGCUUGGU UG#AGCGAUUC   1440UACAUGCCUG UUCGCGGUAC GGUUUUGCUG GAUGGCCAUG ACAUCAAGGA CC#UCAAUCUC   1500CGCUGGCUUC GCCAACAGAU CUCUUUGGUU AGCCAGGAGC CUGUUCUUUU UG#GCACGACG   1560AUUUAUAAGA AUAUUAGGCA CGGUCUCAUC GGCACAAAGU ACGAGAAUGA AU#CCGAGGAU   1620AAGGUCCGGG AACUCAUCGA GAACGCGGCA AAAAUGGCGA AUGCUCAUGA CU#UUAUUACU   1680GCCUUGCCUG AAGGUUAUGA GACCAAUGUU GGGCAGCGUG GCUUUCUCCU UU#CAGGUGGC   1740CAGAAACAGC GCAUUGCAAU CGCCCGUGCC GUUGUUAGUG ACCCAAAAAU CC#UGCUCCUG   1800GAUGAAGCUA CUUCGGCCUU GGACACAAAA UCCGAAGGCG UGGUUCAAGC AG#CUUUGGAG   1860AGGGCAGCUG AAGGCCGAAC UACUAUUGUG AUCGCUCAUC GCCUUUCCAC GA#UCAAAACG   1920GCGCACAACA UUGUGGUUCU GGUCAAUGGC AAAAUUGCUG AACAAGGAAC UC#ACGAUGAA   1980UUGGUUGACC GCGGAGGCGC UUAUCGCAAA CUUGUGGAGG CUCAACGUAU CA#AUGAACAG   2040AAGGAAGCUG ACGCCUUGGA GGACGCCGAC GCUGAGGAUC UCACGAAUGC AG#AUAUUGCC   2100AAAAUCAAAA CUGCGUCAAG CGCAUCAUCC GAUCUCGACG GAAAACCCAC AA#CCAUUGAC   2160CGCACGGGCA CCCACAAGUC UGUUUCCAGC GCGAUUCUUU CUAAAAGACC CC#CCGAAACA   2220ACUCCGAAAU ACUCAUUAUG GACGCUGCUC AAAUUUGUUG CUUCCUUCAA CC#GCCCUGAA   2280AUCCCGUACA UGCUCAUCGG UCUUGUCUUC UCAGUGUUAG CUGGUGGUGG CC#AACCCACG   2340CAAGCAGUGC UAUAUGCUAA AGCCAUCAGC ACACUCUCGC UCCCAGAAUC AC#AAUAUAGC   2400AAGCUUCGAC AUGAUGCGGA UUUCUGGUCA UUGAUGUUCU UCGUGGUUGG UA#UCAUUCAG   2460UUUAUCACGC AGUCAACCAA UGGUGCUGCA UUUGCCGUAU GCUCCGAGAG AC#UUAUUCGU   2520CGCGCGAGAA GCACUGCCUU UCGGACGAUA CUCCGUCAAG ACAUUGCUUU CU#UUGACAAG   2580GAAGAGAAUA GCACCGGCGC UCUGACCUCU UUCCUGUCCA CCGAGACGAA GC#AUCUCUCC   2640GGUGUUAGCG GUGUGACUCU AGGCACGAUC UUGAUGACCU CCACGACCCU AG#GAGCGGCU   2700AUCAUUAUUG CCCUGGCGAU UGGGUGGAAA UUGGCCUUAG UUUGUAUCUC GG#UUGUGCCG   2760GUUCUCCUGG CAUGCGGUUU CUACCGAUUC UAUAUGCUAG CCCAGUUUCA AU#CACGCUCC   2820AAGCUUGCUU AUGAGGGAUC UGCAAACUUU GCUUGCGAGG CUACAUCGUC UA#UCCGCACA   2880GUUGCGUCAU UAACCCGGGA AAGGGAUGUC UGGGAGAUUU ACCAUGCCCA GC#UUGACGCA   2940CAAGGCAGGA CCAGUCUAAU CUCUGUCUUG AGGUCAUCCC UGUUAUAUGC GU#CGUCGCAG   3000GCACUUGUUU UCUUCUGCGU UGCGCUCGGG UUUUGGUACG GAGGGACACU UC#UUGGUCAC   3060CACGAGUAUG ACAUUUUCCG CUUCUUUGUU UGUUUCUCCG AGAUUCUCUU UG#GUGCUCAA   3120UCCGCGGGCA CCGUCUUUUC CUUUGCACCA GACAUGGGCA AGGCGAAGAA UG#CGGCCGCC   3180GAAUUCCGAC GACUGUUCGA CCGAAAGCCA CAAAUUGAUA ACUGGUCUGA AG#AGGGCGAG   3240AAGCUCGAAA CGGUGGAAGG UGAAAUCGAA UUUAGGAACG UGCACUUCAG AU#ACCCGACC   3300CGCCCAGAAC AGCCUGUCCU GCGCGGCUUG GACCUGACCG UGAAGCCUGG AC#AAUAUGUU   3360GCGCUUGUCG GACCCAGCGG UUGUGGCAAG AGUACCACCA UUGCAUUGCU UG#AGCGCUUU   3420UACGAUGCGA UUGCCGGGUC CAUCCUUGUU GAUGGGAAGG ACAUAAGUAA AC#UAAAUAUC   3480AACUCCUACC GCAGCUUUCU GUCACUGGUC AGCCAGGAGC CGACACUGUA CC#AGGGCACC   3540AUCAAGGAAA ACAUCUUACU UGGUAUUGUC GAAGAUGACG UACCGGAAGA AU#UCUUGAUU   3600AAGGCUUGCA AGGACGCUAA UAUCUACGAC UUCAUCAUGU CGCUCCCGGA GG#GCUUUAAU   3660ACAGUUGUUG GCAGCAAGGG AGGCAUGUUG UCUGGCGGCC AAAAGCAACG UG#UGGCCAUU   3720GCCCGAGCCC UUCUUCGGGA UCCCAAAAUC CUUCUUCUCG AUGAAGCGAC GU#CAGCCCUC   3780GACUCCGAGU CAGAAAAGGU CGUCCAGGCG GCUUUGGAUG CCGCUGCCCG AG#GCCGAACC   3840ACAAUCGCCG UUGCACACCG ACUCAGCACG AUUCAAAAGG CGGACGUUAU CU#AUGUUUUC   3900GACCAAGGCA AGAUCGUCGA AAGCGGAACG CACAGCGAAC UGGUCCAGAA AA#AGGGCCGG   3960 UACUACGAGC UGGUCAACUU GCAGAGCUUG GGCAAGGGCC AU    #                   #4002

I claim:
 1. A method for determining the fungal multiple drug resistance (MDR) inhibition activity of a compound which comprises: a) placing a culture of fungal cells, transformed with a vector which expresses a nucleic acid encoding an atrD protein consisting essentially of the amino acid sequence of SEQ ID NO:2, in the presence of: (i) an antifungal agent to which said fungal cell is resistant, but to which said fungal cell is sensitive in its untransformed state; (ii) a compound suspected of possessing Aspergillus nidulans MDR inhibition activity; and b) determining the fungal MDR inhibition activity of said compound by measuring the ability of the antifungal agent to inhibit the growth of said fungal cell.
 2. The method of claim 1 wherein the fungal cell is Saccharomyces cerevisiae.
 3. The method of claim 1 wherein the culture of fungai cells is transformed with a vector which expresses a nucleic acid encoding an atrD protein of the amino acid sequence of SEQ ID NO:2. 